Mapping charge to function relationships of the DNA mimic protein Ocr

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Abstract

This thesis investigates the functional consequences of neutralising the negative charges on
the bacteriophage T7 antirestriction protein ocr. The ocr molecule is a small highly
negatively charged, protein homodimer that mimics a short DNA duplex upon binding to the
Type I Restriction Modification (RM) system. Thus, ocr facilitates phage infection by
binding to and inactivating the host RM system.
The aim of this study was to analyse the effect of reducing the negative charge on the ocr
molecule by mutating the acidic residues of the protein. The ocr molecule (117 residues) is
replete with Asp and Glu residues; each monomer of the homodimer contains 34 acidic
residues. Our strategy was to begin with a synthetic gene in which all the acidic residues of
ocr had been neutralised. This so called ‘positive ocr’ (or pocr) was used as a template to
gradually reintroduce codons for acidic residues by adapting the ISOR strategy proposed by
D.S.Tawfik. After each round of mutagenesis an average of 4-6 acidic residues were
incorporated into pocr. In this fashion a series of mutant libraries in which acidic residues
were progressively introduced into pocr was generated.
A high-throughput in vivo selection assay was developed and validated by assessing the
antirestriction behaviour of a number of mutants of the DNA mimic proteins wtOcr and
Orf18 ArdA. Further to this, selective screening of the libraries allowed us to select clones
that displayed antirestriction activity. These mutants were purified and in vitro
characterisation confirmed these mutants as displaying the minimum number of acidic
residues deemed critical for the activity of ocr. This in vitro process effectively simulated the
evolution of the charge mimicry of ocr.
Moreover, we were able to tune the high-throughput assay to different selection criteria in
order to elucidate various levels of functionality and unexpected changes in phenotype. This
approach enables us to map the “in vitro” evolution of ocr to identify acidic residues that are
required for protein expression, solubility and function proceeding to a fully functional
antirestriction protein.